Supportive or detrimental roles of P2Y receptors in brain pathology?—The two faces of P2Y receptors in stroke and neurodegeneration detected in neural cell and in animal model studies

This review describing the role of P2Y receptors in neuropathological conditions focuses on obvious differences between results demonstrating either a role in neuroprotection or in neurodegeneration, depending on in vitro and in vivo models. Such critical juxtaposition puts special emphasis on discussions of beneficial and detrimental effects of P2Y receptor agonists and antagonists in these models. The mechanisms reported to underlie the protection in vitro include increased expression of oxidoreductase genes, like carbonyl reductase and thioredoxin reductase; increased expression of inhibitor of apoptosis protein-2; extracellular signal-regulated kinase- and Akt-mediated antiapoptotic signaling; increased expression of Bcl-2 proteins, neurotrophins, neuropeptides, and growth factors; decreased Bax expression; non-amyloidogenic APP shedding; and increased neurite outgrowth in neuronal cells. Animal studies investigating the influence of P2Y receptors in middle cerebral artery occlusion (MCAO) models for stroke prove beneficial effects of P2Y receptor antagonists. In MCAO mice and rats, the application of broad-range P2 receptor antagonists decreased the infarct volume and improved neurological outcome. Moreover, antagonists of the P2Y₁ receptor, one of the most abundant P2Y receptor subtypes in brain tissue, decreased neuronal loss and improved spatial memory in rats after traumatic brain injury (TBI). Currently available data show a discrepancy between in vitro and in vivo models concerning the benefits of P2Y receptor activation in pathological conditions. In vitro models demonstrate protection by P2Y receptor agonists, but in vivo P2Y receptor activation deteriorates the outcome after MCAO and controlled cortical impact brain injury, a TBI model. To broaden the scope of the review, we additionally discuss publications that demonstrate detrimental effects of P2Y receptor agonists in vitro and publications showing protective effects of agonists in vivo. All these studies help to better understand the significant role of P2Y receptors especially in stroke models and to develop pharmacological strategies for the treatment of stroke.     

Autocrine regulation of γ-irradiation-induced DNA damage response via extracellular nucleotides-mediated activation of P2Y6 and P2Y12 receptors

A key component of the response to DNA damage caused by ionizing radiation is DNA repair. Release of extracellular nucleotides, such as ATP, from cells plays a role in signaling via P2 receptors. We show here that release of ATP, followed by activation of P2Y receptors, is involved in the response to γ-irradiation-induced DNA damage. Formation of phosphorylated histone variant H2AX (γH2AX) foci, which are induced in nuclei by DNA damage and contribute to accumulation of DNA-repair factors, was increased at 1-3h after γ-ray irradiation (2.0Gy) of human lung cancer A549 cells. Focus formation was suppressed by pre-treatment with the ecto-nucleotidase apyrase. Pre-treatment with ecto-nucleotidase inhibitor ARL67156 or post-treatment with ATP or UTP facilitated induction of γH2AX, indicating that extracellular nucleotides play a role in induction of γH2AX foci. Next, we examined the effect of P2 receptor inhibitors on activation of ataxia telangiectasia mutated (ATM; a protein kinase) and accumulation of 53BP1 (a DNA repair factor), both of which are important for DNA repair, at DNA damage sites. P2Y6 receptor antagonist MRS2578, P2Y12 receptor antagonist clopidogrel, and P2X7 receptor antagonists A438079 and oxATP significantly inhibited these processes. Release of ATP was detected within 2.5min after irradiation, but was blocked by A438079. Activation of ATM and accumulation of 53BP1 were decreased in P2Y6 or P2Y12 receptor-knockdown cells. We conclude that autocrine/paracrine signaling through P2X7-dependent ATP release and activation of P2Y6 and P2Y12 receptors serves to amplify the cellular response to DNA damage caused by γ-irradiation.     

The role of the P2X₇ receptor in infectious diseases

ATP is an extracellular signal for the immune system, particularly during an inflammatory response. It is sensed by the P2X₇ receptor, the expression of which is upregulated by pro-inflammatory cytokines. Activation of the P2X₇ receptor opens a cation-specific channel that alters the ionic environment of the cell, activating several pathways, including (i) the inflammasome, leading to production of IL-1β and IL-18; (ii) the stress-activated protein kinase pathway, resulting in apoptosis; (iii) the mitogen-activated protein kinase pathway, leading to generation of reactive oxygen and nitrogen intermediates; and (iv) phospholipase D, stimulating phagosome-lysosome fusion. The P2X₇ receptor can initiate host mechanisms to remove pathogens, most particularly those that parasitise macrophages. At the same time, the P2X₇ receptor may be subverted by pathogens to modulate host responses. Moreover, recent genetic studies have demonstrated significant associations between susceptibility or resistance to parasites and bacteria, and loss-of-function or gain-of-function polymorphisms in the P2X₇ receptor, underscoring its importance in infectious disease.     

The role of γ-aminobutyric acid and its receptors in the nucleus of basal optic root in pigeons

The effects of γ-aminobutyric acid (GABA) and its antagonists bicuculline and 2-hydroxysaclofen on neuronal firings in the nucleus of basal optic root (nBOR) in pigeons were studied by using extracellular recording and microiontophoretic techniques. The results suggest that GABA may be an inhibitory neurotransmitter or modulator within nBOR, functioning by means of main mediation of GABAA receptors and of minor mediation of GABAB receptors. Furthermore, GABA and its GABAA receptors are involved in the modulation of directional selectivity in part of nBOR neurons.     

The role of TRPM2 in pancreatic β-cells and the development of diabetes

TRPM2 is a Ca²⁺-permeable non-selective cation channel that can be activated by adenosine dinucleotides, hydrogen peroxide, or intracellular Ca²⁺. The protein is expressed in a wide variety of cells, including neurons in the brain, immune cells, endocrine cells, and endothelial cells. This channel is also well expressed in β-cells in the pancreas. Insulin secretion from pancreatic β-cells is the primary mechanism by which the concentration of blood glucose is reduced. Thus, impairment of insulin secretion leads to hyperglycemia and eventually causes diabetes. Glucose is the principal stimulator of insulin secretion. The primary pathway involved in glucose-stimulated insulin secretion is the ATP-sensitive K⁺ (K_ATP) channel to voltage-gated Ca²⁺ channel (VGCC)-mediated pathway. Increases in the intracellular Ca²⁺ concentration are necessary for insulin secretion, but VGCC is not sufficient to explain [Ca²⁺]_i increases in pancreatic β-cells and the resultant secretion of insulin. In this review, we focus on TRPM2 as a candidate for a [Ca²⁺]_i modulator in pancreatic β-cells and its involvement in insulin secretion and development of diabetes. Although further analyses are needed to clarify the mechanism underlying TRPM2-mediated insulin secretion, TRPM2 could be a key player in the regulation of insulin secretion and could represent a new target for diabetes therapy.     

Opposite effects of P2X7 and P2Y2 nucleotide receptors on α-secretase-dependent APP processing in Neuro-2a cells

The amyloid precursor protein (APP) is proteolytically processed by β- and γ-secretases to release amyloid-β peptide (Aβ), the main component found in senile plaques of Alzheimer's disease (AD) patient brains. Alternatively, APP can be cleaved within the Aβ sequence by α-secretase, thus precluding the generation of Aβ. We have demonstrated that activation of the P2X7 receptor leads to a reduction of α-secretase activity in Neuro-2a cells. Moreover, the P2X7 ligand 2'(3')-O-(4-benzoylbenzoyl) ATP (BzATP) can also activate a different P2 receptor in these cells. This receptor, whose pharmacology resembles that of the P2Y(2) receptor, has an opposite effect, leading to increases in α-secretase activity. Our study suggests that P2X7R and P2Y(2)R could be novel therapeutic targets in AD.     

The αMβ2 integrin and its role in neutrophil function

Neutrophils are the first cell type to arrive at the injury sites and play a critical role in host defense,by virtue of its ability to adhere and transmigrate through endothelium,to phagocytose foreign pathogens,and to produce free oxygen radicals and proteolytic enzymes.Yet,inappropriate neutrophil activation causes tissue damage and various inflammatory diseases.These physiological and pathological functions of neutrophils depend on the engagement of certain surface receptors,especially αMβ2,the major β2 integrin receptor present on neutrophil surface.Understanding of the molecular mechanisms underlying ligand binding by αMβ2,as well as the rolea of αMβ2ligand interactions in neutrophil functions will enable us to regulate more precisely neutrophil activities:that is,to promote their host defense functions,and at the same time to minimize their deleterious effects of normal cells.     

Structure–activity relationships of dinucleotides: Potent and selective agonists of P2Y receptors

Dinucleoside polyphosphates act as agonists on purinergic P2Y receptors to mediate a variety of cellular processes. Symmetrical, naturally occurring purine dinucleotides are found in most living cells and their actions are generally known. Unsymmetrical purine dinucleotides and all pyrimidine containing dinucleotides, however, are not as common and therefore their actions are not well understood. To carry out a thorough examination of the activities and specificities of these dinucleotides, a robust method of synthesis was developed to allow manipulation of either nucleoside of the dinucleotide as well as the phosphate chain lengths. Adenosine containing dinucleotides exhibit some level of activity on P2Y₁ while uridine containing dinucleotides have some level of agonist response on P2Y₂ and P2Y₆. The length of the linking phosphate chain determines a different specificity; diphosphates are most accurately mimicked by dinucleoside triphosphates and triphosphates most resemble dinucleoside tetraphosphates. The pharmacological activities and relative metabolic stabilities of these dinucleotides are reported with their potential therapeutic applications being discussed.     

The role of AMPKα2 in the HFD-induced nonalcoholic steatohepatitis

Nonalcoholic fatty liver disease (NAFLD) is associated with hepatic steatosis, inflammation and liver fibrosis and has become one of the leading causes of hepatocellular carcinoma and liver failure. However, the underlying molecular mechanism of hepatic steatosis and the progression to nonalcoholic steatohepatitis (NASH) are not fully understood. Herein, we discovered that AMPKα2 catalytic subunit showed reduced expression in the liver following high fat diet (HFD) feeding to mice. Importantly, knockout of AMPKα2 in mice aggravated NAFLD, hepatic steatosis, inflammation and fibrosis. On the other hand, hepatocyte-targeted overexpression of AMPKα2 prevented or reversed NAFLD indications. In vivo mechanistic studies revealed that increased phosphorylation of IKKα/β and NF-κB in HFD-fed AMPKα2^−/− mice compared to WT mice, and treatment of these mouse cohorts with an inhibitor of NF-κB signaling for 4 weeks, effectively attenuated the progression of steatohepatitis and metabolic disorder features. In summary, AMPKα2 provides a protective role in the process of hepatic steatosis to NASH progression through suppression of liver NF-κB signaling.     

The role of 11β-hydroxysteroid dehydrogenase type 2 in human hypertension

Cortisol and aldosterone have the same in vitro affinity for the mineralocorticoid receptor (MR), although in vivo only aldosterone acts as a physiologic agonist of the MR, despite circulating levels of cortisol in humans and corticosterone in rodents being three orders of magnitude higher than aldosterone levels. In mineralocorticoid target organs the enzyme 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2) inactivates 11-hydroxy steroids, to their inactive keto-forms, thus protecting the nonselective MR from activation by glucocorticoids. The gene is highly expressed in all sodium-transporting epithelia, particularly in the kidney and colon, but also in human placenta and vascular wall. Mutations in the HSD11B2 gene cause a rare monogenic juvenile hypertensive syndrome called apparent mineralocorticoid excess (AME). In AME, compromised 11βHSD2 enzyme activity results in activation of the MR by cortisol, causing sodium retention, hypokalaemia, and salt-dependent hypertension. Whereas mutations or inhibition of 11βHSD2 by licorice have been clearly shown to produce a congenital or acquired syndrome of mineralocorticoid excess, the questions remaining are the extent to which subtle abnormalities in MR/11βHSD2 mechanisms may contribute to essential hypertension. Studies in patients with essential hypertension showed a prolonged half-life of cortisol and an increased ratio of urinary cortisol to cortisone metabolites, suggesting a deficient 11βHSD2 activity. These abnormalities may be genetically determined, as suggested by the association of a microsatellite flanking the HSD11B2 gene with hypertension in black patients with end-stage kidney disease and with salt sensitivity of blood pressure in healthy subjects. These findings indicate that variants of the HSD11B2 gene may contribute to the enhanced blood pressure response to salt and possibly to hypertension in humans.     

The role of PKC isoforms in the inhibition of NF-κB activation by vitamin K2 in human hepatocellular carcinoma cells

Vitamin K (VK) has diverse protective effects against osteoporosis, atherosclerosis and carcinogenesis. We recently reported that menatetrenone, a VK2 analogue, suppressed nuclear factor (NF)-κB activation in human hepatoma cells. Although NF-κB is regulated by isoforms of protein kinase C (PKC), the involvement of PKCs in VK2-mediated NF-κB inhibition remains unknown. Therefore, the effects of VK2 on the activation and the kinase activity of each PKC isoform were investigated. The human hepatoma Huh7 cells were treated with PKC isoform-specific inhibitors and/or siRNAs against each PKC isoform with or without 12-O-tetradecanoylphorbol-13-acetate (TPA). VK2 inhibited the TPA-induced NF-κB activation in Huh7 cells. NF-κB activity was inhibited by the pan-PKC inhibitor Ro-31-8425, but not by the PKCα-specific inhibitor Gö6976. The knockdown of individual PKC isoforms including PKCα, δ and ? showed only marginal effects on the NF-κB activity. However, the knockdown of both PKCδ and PKC?, together with treatment with a PKCα-specific inhibitor, depressed the NF-κB activity. VK2 suppressed the PKCα kinase activity and the phosphorylation of PKC? after TPA treatment, but neither the activation nor the enzyme activity of PKCδ was affected. The knockdown of PKC? abolished the TPA-induced phosphorylation of PKD1, and the effects of PKD1 knockdown on NF-κB activation were similar to those of PKC? knockdown. Collectively, all of the PKCs, including α, δ and ?, and PKD1 are involved in the TPA-mediated activation of NF-κB. VK2 inhibited the NF-κB activation through the inhibition of PKCα and ? kinase activities, as well as subsequent inhibition of PKD1 activation.     

The inductive role of Wnt-β-Catenin signaling in the formation of oral apparatus

Proper patterning and growth of oral structures including teeth, tongue, and palate rely on epithelial–mesenchymal interactions involving coordinated regulation of signal transduction. Understanding molecular mechanisms underpinning oral–facial development will provide novel insights into the etiology of common congenital defects such as cleft palate. In this study, we report that ablating Wnt signaling in the oral epithelium blocks the formation of palatal rugae, which are a set of specialized ectodermal appendages serving as Shh signaling centers during development and niches for sensory cells and possibly neural crest related stem cells in adults. Lack of rugae is also associated with retarded anteroposterior extension of the hard palate and precocious mid-line fusion. These data implicate an obligatory role for canonical Wnt signaling in rugae development. Based on this complex phenotype, we propose that the sequential addition of rugae and its morphogen Shh, is intrinsically coupled to the elongation of the hard palate, and is critical for modulating the growth orientation of palatal shelves. In addition, we observe a unique cleft palate phenotype at the anterior end of the secondary palate, which is likely caused by the severely underdeveloped primary palate in these mutants. Last but not least, we also discover that both Wnt and Shh signalings are essential for tongue development. We provide genetic evidence that disruption of either signaling pathway results in severe microglossia. Altogether, we demonstrate a dynamic role for Wnt-β-Catenin signaling in the development of the oral apparatus.     

The role of ERK and Smad2 signal pathways in the alternatively activated macrophages induced by TGF-β1 and high-ambient glucose

Macrophages can be alternatively activated by TGF-β1 and high-ambient glucose, in which the role of Smad2 and the crosstalk between ERK and Smad2 pathways are not fully understood. The activation of ERK and Smad2 pathways and the expression of arginase-1 were detected by Western blot. The role of Smad2 and the relationship between ERK and Smad2 pathways were investigated by using biochemical inhibitors. The protein of arginase-1 was significantly overexpressed in RAW264.7 cells stimulated by TGF-β1 and high-ambient glucose, which can be partially blocked by not only U0126 (ERK inhibitor) but also SB431542 (Smad2 inhibitor). Furthermore, simply inhibiting one pathway had no effect on the other pathway. In conclusion, both ERK and Smad2 signal pathways are involved in the activation of macrophages induced by TGF-β1 and high-ambient glucose, while there is no crosstalk shown in the process.     

The localization of the β-subtype of protein kinase C (PKC-β) in rat sympathetic neurons

Summary The localization of PKC- was studied in rat sympathetic neurons using a polyclonal antibody specific for the ₁- and ₂-subspecies. The tissues studied included the superior cervical (SCG) and hypogastric (HGG) ganglia and the target tissues of the SCG and HGG neurons: the submandibular gland, iris, prostate and vas deferens. PKC--LI was found in nerve fibers in both ganglia. A proportion of the fibers in the SCG disappeared after decentralization, suggesting that the fibers were of both pre- and postganglionic origin. The somata of the HGG and SCG neurons expressed varying amounts of PKC--LI, the majority of SCG neurons being labelled only after colchicine treatment. In all target tissues there were PKC--immunoreactive nerve fibers in bundles, but the most peripheral branches of the fibers were negatively labelled. The results show that PKC--LI is widely present in sympathetic postganglionic neurons with mainly quantitative differences. The lack of PKC- in the most peripheral branches of nerve fibers might be a general feature of sympathetic postganglionic neurons, suggesting that the participation of PKC- in neurotransmitter release and in other functions in nerve terminals in sympathetic adrenergic neurons is unlikely.